CN102770555B - Improved bacterial membrane protein secrection - Google Patents

Abstract

Improved bacterial secretion signals derived from pelB and ompA are provided. The improved variants enhance bacterial membrane secretion are thus useful for production of proteins secreted from bacteria including proteins displayed on filamentous phage particles, and, in particular, proteins requiring oxidative formation of covalent bonds, such as disulfide bonds within or between polypeptide chains in order to form a correctly folded and functional protein structure. Described herein are methods for the multivalent display of complex dimeric proteins on the surface of a bacteriophage particle and combinatorial synthetic libraries of such proteins displayed as a fusion polypeptide with filamentous phage pIX coat protein. Heterodimeric or more complex interchain bonded structures may also be displayed using the method of the invention.

Description

The bacterial membrane protein secretion improved

Background technology

priority application

This application claims the U. S. application No.61/261 submitted on November 17th, 2009, the right of priority of 768, this patent is incorporated to herein by reference in full.

Technical field

The present invention relates to the modification method making protein secreting pass bacterium plasma membrane, the method comprise filamentous phage coat protein is used for show exogenous protein comprise can for monomer, dimeric or heterodimer or polymeric protein (comprises complete antibody or antibody fragment, or the polymer construct of other disulfide linkage link) the library of variant, the method realizes by saltant type pelB or saltant type ompA secretion signal being connected with described foreign protein (exoprotein) construct.

about the discussion of this area

Filamentous phage display is a kind of widely used technology selecting protein based on avidity, because the N of the nucleic acid of coded polypeptide and its coat protein holds fusion to link together by each phage particle in the selection process.M13 phage encoded five kinds of coat protein, have secondary coat protein pIII and the pVI of general five copies, have pVII and pIX of similar number at the other end of phage in one end of phage.Phage DNA is encapsulated by the major cat protein pVIII of general 3000 copies.Although achieve the displaying of allogenic polypeptide by often kind of coat protein of M13, but pIII and pVIII is still fusion partners the most frequently used at present.Use this technology, constructed peptide, Fab, scFv and other protein conjugates (protein binder) library and can be used for various application, there is immense commercial value.

Due to the length of pIII coat protein and conformation, it is comparatively more welcome in other protein.The secondary coat protein of pIII is the protein that responsible phage-infect enters 402 amino acid in intestinal bacteria, 42kD, and it comprises two structural domains connected by flexible hinge.Hold with pIII N the protein shown merging and can hitch and leave phage particle, thus decrease the ability of its pIII function needed for interference and make part can enough close to combining further.By contrast, pVII and pIX is respectively 33 and 32 amino acid whose short coilin matter, in phage surface dense packing.But, report is had to describe the displaying (Gao of scFv library on pIX, C. people is waited, Proc Natl Acad Sci U S A 99,12612-12616,2002) with by utilizing the ability making not homopolypeptide and pVII and closely adjacent both pIX merge to carry out Heterodimeric body display people such as (, 1999 Proc Nat Acad Sci 96:6025-6030 and Janda US7078166) Gao to Fv or Fab library.Show that the phage of pVII fusions also has been reported people such as (, 2005.J Immunol Methods 307:135) Kwasnikowski.Use hybridization phage vector or phagemid vector, the peptide, Fab and other protein (WO2009/085462, WO2009/085464 and WO2009/085468) that merge with pIX coat protein can be shown on phage.Also have been described a kind of alternative method, the foreign protein (exoprotein) of being encoded by phage or phagemid vector does not in the method merge with coat protein, but is covalently attached to again engineered coat protein pIII and pIX (US6753136) by disulfide bonding.

Intestinal bacteria are one of host being widely used in generation recombinant protein most.But, often have problems in the most of output reclaiming correct folding protein.A kind of method addressed these problems is that recombinant protein is secreted in periplasmic space or substratum.Secretion is prepared recombinant protein and is had some advantages, and as simplified purifying, avoiding protease attack and N to hold Met to extend, and the probability that protein correctly folds is better.

At the DNA replication dna of phage protein with after expressing, M13 and other filobactiviruses are assembled at host cell membrane place.It is the committed step that phage is assembled that phage structural protein are transported in film.In the practice of phage display, bacterial signal peptide is widely used in construct and has helped fusion rotein and be transported through film, and be that the displaying of the fusion rotein realizing some type is crucial.Signal peptide from the pectin lyase B (pelB) of carrot soft rot Erwinia (Erswinia carotovora) has been widely used in strengthen to use bacterial cell generation protein and strengthen and has produced both protein in phage display.

Utilize bacterial host cell and with library form of recombinating, can carry out carrying out in selection being favourable with again engineered complex proteins structure to modified by microbial culture or in protein such as the ability of protein dimer that phage particle displaying on surface is more various and complicated.Need to advance prior art to produce the high throughput method of the variant of highly effective method of producing protein and screening complex proteins always, the variant of described complex proteins is the variant of such as human IgG, and human IgG is that the heavy chain and light chain that are connected by intermolecular disulfide linkage are to the homodimer of (heterodimer).

Summary of the invention

The invention provides in bacterium plasma membrane, (be such as the phage particle assembled in host bacterium) for exogenous protein and express, the modification method of transhipment and assembling, the method realizes with the nucleic acid of the encode mutant peIB signal sequence of the nucleic acid effective integration of the aminoacid sequence expecting peptide or the peptide sequence expressed and/or show of encoding by mixing, this saltant type pelB signal has sequence MKYLLSTAAAGLLLLAAQPAMA, is the P6S variant of (SEQ ID NO:1).In one aspect of the invention, the saltant type SEQ ID NO:2 of the sequence of encode mutant pelB signal can be the base of wherein position 16 be thymidine.In another embodiment of the present invention, coded secretion signal is the saltant type ompA with sequence MKKTAIAIAVPLAGFATVAQA, and it is the A11P variant (SEQ ID NO:3) of wild-type sequence.In one aspect, the saltant type SEQ ID NO:4 of the sequence of encode mutant ompA secretion signal can be the broken base at wherein position 31 place be cytidine(C.In the method for the invention, the secretion signal P6S of SEQ ID NO:1 is used for strengthening the transhipment of protein in bacterial cell culture.

The invention provides for the modification method of protein multivalent display on phage particle and the means of showing dimer or the polymer protein merged with bacteriophage coat protein on phage particle.In one embodiment, multivalent display is the multivalent display holding the foreign protein merged with the N of the secondary coat protein of phage pVII or pIX.The present invention also contain two kinds of different foreign proteins on phage particle while express and show, wherein the first albumen and pVII merge and the second albumen and pIX merge, and this is by merging to realize by the sequence of P6S variant (SEQ ID NO:1) of encoding secretion signals pelB and the foreign protein coding region of pIX or pVII fusion rotein (it is phage or phagemid vector).In one aspect, the polypeptide merged with pVII or pIX can form interchain and combine on phage, form more higher structure, the protein that the protein connected as dimer or polymeric pVII or dimer or polymeric pIX connect, or wherein one or more foreign proteins merge from a bacteriophage coat protein and the dimer that merges of identical or different foreign protein and different bacteriophage coat proteins or multimeric structure.Form disulfide linkage between the polypeptide chain that polymer protein can merge at bacteriophage coat protein, or form more complicated structure by combining with the foreign protein of absolute coding and expression.In an example of the dimeric structure of disulfide bonding, the protein formed comprises the antibody Fc domain of the structural domain with interchain disulfide bond and intrachain disulfide bond bonding, and this antibody Fc domain can be combined with Fc acceptor, albumin A or complement factor C1q.In one aspect, also combine with light chain of antibody to form total length IgG molecule by with the protein structure that the coat protein that polypeptide chain merges is formed.In another embodiment, article two, different polypeptide display is pVII and pIX fusions, and wherein said polypeptide can form interchain that is non-covalent or covalency character and combine (association) to form dimer or polymer protein structure on filobactivirus.

The growth that the phage that the invention provides the foreign protein that expression is merged with the coat protein being selected from pIII, pVII, pVIII or pIX increases in culture, this is realized by the sequence of the peptide mixing the secretion signal coding region being selected from SEQ ID NO:2 and the 4 or SEQ ID NO:1 or 3 that encodes merged with described foreign protein coding region.

The invention provides a kind of replicable vector, its coding at least one fusion rotein, have the nucleotide sequence of encoding exogenous polypeptide merged with the sequence that is selected from the bacteriophage coat protein of pIII, pVII, pVIII or pIX of encoding, wherein the secretion signal encoding sequence of the P6S variant of coding SEQ ID NO:1 or the A10P variant of SEQ ID NO:3 and described foreign protein encoding sequence merge.In one embodiment of the invention, the carrier of encoding said proteins bacteriophage coat protein fusions is included in coding and maybe can be carried out by the protein tag of part or antibody recognition, protease cracking site or static electrification district the other sequence inserted between the protein in chemically conjugated region (as lysine residue).In one aspect, described other sequence is HA sequence (SEQ ID NO:5), Flag (SEQ ID NO:6) district or six Histidines (His6, SEQ ID NO:7).

Use secretion signal of the present invention another in, secretion signal is used for build phage or phagemid vector, wherein there is diversified district (variegated region) in heterologous protein coding sequence outside, for the library showing variant proteins.In a preferred embodiment, the foreign protein part of fusion rotein is antibody or its fragment, and the library so formed is antibody or antibody fragment (as scFv or Fab) library.In one aspect of the invention, variant secretion signal is used for into the construct library of the polypeptide chain (as Fc fusion rotein) of homodimer.In another embodiment, homodimeric body structure can be connected to form more complicated structure with special-shaped polypeptide further.In one aspect, the exhibition both antibody heavy chain polypeptide and light chain polypeptide in single phage molecule causes functional antibody molecule surface-mounted at phage particle, such as but not limited to complete IgG molecule.Comprise in the present invention be containing replicable vector and the host cell that can be the phage particle of the protein dimer that disulfide linkage is connected in its surface display fusion polypeptide.The library of generation like this can show can be used for assemble, screening and such as this area implement for the selection and improvement and so on of antibody compositions other query property technology (interrogative technique) from the beginning library.

Accompanying drawing explanation

Figure 1A-B is the diagram (A) of clone's box for expressing pVII fusion rotein and the schematic diagram (B) for clone's box of the double expression(DE) of pVII and pIX fusion rotein.

Fig. 2 A-E shows about the specific binding of antibody with the phage of displaying HA (A), FLAG (B), His-6 (C), and about the result of solubility dimer EGFR-Ig construct with the Phage-ELISA of the specific binding of the specific binding of phage and the phage of Streptavidin and displaying PHPEP 97 (E) of showing PHPEP 190 (D); Wherein M13KE phage is used as negative control.

Fig. 3 shows the result with the ELISA on the phage being derived from HA-pVII phage vector (Figure 1A) compared with the displaying on hybrid vector (for the HA shown and pIX is connected) derivative phage, wherein anti-HA antibody is used for specific detection, and anti-flag antibody is used as the control antibodies of experiment.

Fig. 4 shows the displaying of HA peptide on the phage coming from the pVII phage vector (pelB-Ha-pVII) with pelB signal sequence or the pVII phage vector (HA-pVII) without pelB signal sequence.Anti-flag antibody is used as control antibodies in this experiment.

Fig. 5 shows the sequence of the signal peptide for this research, and has in the recombinant phage in the generation of normal plaque size and Seedling height titre the position of the sudden change that the monamino acid determined in the sequence causing each expression changes later: on the position 6 of pelB, the Pro Ala sported on the position 11 of Ser (A) and ompA sports Pro (B).

Fig. 6 A-B is from the activity (A) of the alkaline phosphatase (AP) of the bacterial cultures secretion of cultivating among LB or the assay method of activity (B) being retained in the alkaline phosphatase (AP) in bacterial precipitation, one wherein in four sequences is merged by with encoding sequence: wild-type signal sequence pelB (p3915), ompA (p3913), and the pelB (p3916) of M13 gene III (p3912) and sudden change and ompA (p3914).Measure with AP substrate the total AP expressed in the AP and cytolysis thing secreted in LB substratum and carried out normalization method with cell density.

Fig. 7 illustrates the two Phage-ELISA results of showing of assessment HA and FLAG peptide on pVII and pIX of same phage particle: with anti-FLAG antibody and anti-HA antibody test six FLAG-pVII-HA-pIX phage clones in conjunction with density, HA-pVII, FLAG-pVII and M13KE phage is with comparing.

Fig. 8 shows the titre at six time point places, indicates the improvement taking turns (G0-G10 generation) single clonal growth speed in succeeding generations at continuous print more, has wherein found saltant type ompA signal.

Fig. 9 shows the schematic diagram of the phagemid vector built for expressing total length human IgG, the P6S pelB variant be connected with heavy chain is used as secretion signal by this carrier, and wherein heavy chain C end is merged by joint and pIX coat protein, and the light chain merged with ompA secretion signal is as free expression of polypeptides.

Figure 10 A-D be show produce in ELISA method about using multiple antibody domain to phage is expressed, the EMP-1-Fc construct of expression or phage itself to have specific part, the histogram from the signal of the phage of specified prepared product trapping: (A) anti-Fd (CH1) antibody traps; (B) anti-k antibody; (C) anti-CH2 antibody; (D) anti-CH3 antibody.6-2Fab pIX on phage and the NIg on phage are included as negative control.

Figure 11 is histogram, show and use anti-IL 13 antibody that is purchased and under the condition of the competitive solubility anti-IL 13 monoclonal antibody (there is identical specificity with anti-IL13IgG pIX) of presence or absence (grid-like post bar), the signal about the phage trapped from specified prepared product produced in ELISA method.EMP-1-Fc construct is not in conjunction with the negative control of IL13, is included specific for IL13 on phage 6-2Fab pIX as positive control.

Figure 12 A-B be show produce in ELISA method about in plate by the histogram of signal being purchased the phage that anti-IL 13 antibody traps, wherein add after adding IL 13 antibody competitiveness anti-IL13 antibody (6-2 total length IgG) on the phage of increasing amounts or to IL13 without specific control antibodies (anti-EMMPRIN) (A) and plate in by the histogram of signal being purchased the IL13 that anti-IL13 antibody traps, wherein after adding the anti-IL13 antibody be purchased, add the 6-2Fab on phage.With the addition of anti-il 13 antibodies or the anti-EMMPRIN monoclonal antibody (B) of increasing amounts.

Figure 13 shows after the total length IgG construct being used for biotinylation IL13 or IL17A antigen to trap displaying IL13 or IL17A and when the competitive solubility anti-il 13 antibodies of presence or absence or anti-IL17A monoclonal antibody, carrys out the signal of the phage of any one trapping of the anti-Fd of free domain-specific antibody, anti-k, anti-CH2 and anti-CH3.Phage is detected with anti-M13 antibody (y-axis).

Sequence table is sketched

SEQ ID NO：	Explanation	Feature
			1	Comprise the pelB of variant	P6S
2	The pelB encoding sequence of wild-type and variant	C16T
			3	Comprise the ompA of found variant	A10P
4	The ompA encoding sequence of wild-type and variant	G31C

5	HA
			6	FLAG
7	6xHis
			8	PHPEP 190
9	PHPEP 97

Embodiment

abbreviation

The cytotoxicity of ADCC=antibody dependent cellular mediation, the cytotoxicity of ADMC=antibody dependent monocyte mediation, c1q=complement factor 1q, EPO=recombinant erythropoietin, FcR=Fc acceptor; Ig=immunoglobulin (Ig); Hc=heavy chain; Lc=light chain; IPTG=isopropylthio-beta galactose glycosides; The gene of ompA=encoding E. coli outer membrane protein A; The gene of the pectin lyase gene of pelB=coding carrot soft rot Erwinia

definition

As used herein, except as otherwise noted or based on context apparent, otherwise antibody domain, region and fragment are consistent with standard definition well-known in the art.Protein source of the present invention is from the part of the antibody of one or more immunoglobulin classes or the part of antibody comprising one or more immunoglobulin classes.Immunoglobulin class comprises IgG, IgM, IgA, IgD and IgE isotype, in the situation of IgG and IgA, comprises their hypotype, as IgG ₁, IgG ₂, IgG ₃and IgG ₄.

So-called " cistron " its mean the nucleotide sequence of encoding amino acid sequence in DNA molecular and comprise upstream and downstream DNA and express controlling elements.

So-called " allogenic polypeptide " or " exogenous protein " or " foreign protein " its mean not to be the protein by wild-type filamentous phage genome normal encoding, but be external concerning normal phage protein.Typical allogenic polypeptide is paid close attention to any polypeptide, comprises antibody immunoglobulin heavy chain (Hc) structural domain or light chain immunoglobulin (Lc) structural domain, immunoglobulin heavy chain variable structural domain (V _h), immunoglobulin light chain variable structural domain (V _l), the sequence of polypeptide that is natural or synthesis, single-chain antibody (scFv) or immunoglobulin domains or combination, such as they are particularly as the natural appearance of Fc structural domain that can comprise CH3, CH2, hinge area and/or CH1 structural domain or their fragment.

So-called " Fc " (giving the label of the crystallizable crack fragment of the IgG of papain digestion) its mean the functional fragment of antibody, this fragment comprises the polypeptide chain that is derived from antibody constant structural domain and has the dimeric structure of the disulfide linkage of interchain.In human IgG1, papoid produces C end and (uses as people such as Kabat to Cys226, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. the EU index described in (1991) is numbered, and the document is incorporated to herein by reference clearly) fragment." the EU index as described in Kabat " refers to the residue numbering of human IgG1 EU antibody.Although the definition of the N end residue of Fc may be different, it is generally understood as at least residue 223 comprised in Kabat numbering system, and it is the 3rd residue (C226 in Kabat system) of the halfcystine N end of first inter-chain bonding.The Fc part of this molecule is not directly involved in antibody and contacts with its specific targeted antigen, but mediation effector function.These functions have two types: (1) requires the function that antibody is combined with antigen, combine as C1q and/or CDC (CDC) is active or the Fc receptor y type of IgG combines, the Fc acceptor ε of IgE combines, the Fc receptor alpha of IgA combine after ADCC and ADMC; And (2) do not rely on the function of antigen combination, as the ability by passing biological cells and tissues barrier (as intestines) in conjunction with FCRn and dysuria with lower abdominal colic keeps in the circulating cycle.The ability particularly significantly increasing the serum half-life of antibody molecule or other molecules by the fusion of Fc is highly favourable.Longer molecule of surviving can reduce amount required in clinical treatment, thus reduces the frequency used.

Term " Fc acceptor " or " FcR " are for describing the acceptor be combined with the Fc district of antibody.FcR comprises Fc γ RI, Fc γ RII and Fc γ RIII subclass, comprises the allele variant of these acceptors and the alternative splicing form of these acceptors.Fc γ RII acceptor comprises Fc γ RIIA (" Activating receptor ") and Fc γ RIIB (" Inhibitory receptor "), and they have mainly different at their cytoplasmic domains similar amino acid sequence.Activating receptor Fc γ RIIA contains immunoreceptor tyrosine-based activation motif (FAM) in its cytoplasmic domains.Inhibitory receptor Fc γ RIIB in its cytoplasmic domains containing immunity receptor Tyrosine Inhibitory Motifs (see annu.Rev.Immunol., the summary in 1997,15:203-234; Ravetch and Kinet, Annu.Rev.Immunol., 1991,9:457-92; The people such as Capel, Immunomethods, 1994,4:25-34; And the people such as de Haas, J.Lab.Clin.Med., summarizes FcRs in 1995,126:330-41, is incorporated to by reference by above-mentioned each section of document herein).

So-called " fusion polypeptide " or " fusion rotein " its mean to comprise respectively by the fusion polypeptide (protein) of the first and second polypeptide of the first and second nucleic acid sequence encodings, these two polypeptide are effective connection (fusions).Example as shown here, the fusion rotein that phage particle is shown is the fusions of foreign protein (for the non-natural protein of phage protein group) and bacteriophage coat protein.

The set of the protein coded by term " library " represents, the protein of these codings is variants, and that is, wherein some region is same or similar, and can there be difference in other regions.Variable region can be by determinate variation or random variation (random or nonrandom change).Library or variant can just the number of different variant or " size " in library describe.Available from the beginning antibody library has much higher sample (> 1010), can change, and is easy to set up, and has the background of low undesirably sequence.Library can be accelerated in conjunction with following method set up and low background can be caused: (a) is based on the single-stranded mutagenic of Kunkel; B () has the palindromic loop of restriction site; And (c) uses huge primed method.

" protein dimer " used herein or " polymer protein " refer to more than independently polypeptide or a protein chain, and they are associating and form single spherical protein mutually in vitro or in body.This polymer protein can form to be formed " homodimer " or " homopolymer " by the polypeptide of a more than identical type.Or the polypeptide that this polymer protein also can have unique sequences by more than forms to be formed " heterodimer " or " hetero multimer ".Thus, " hetero multimer " for comprising the molecule of at least the first polypeptide and the second polypeptide, it states the second polypeptide has the difference of at least one amino-acid residue with the first polypeptide on aminoacid sequence.Hetero multimer can comprise " heterodimer " that formed by the first and second polypeptide or can form the more senior quaternary structure wherein existed more than two polypeptide.The example arrangement of hetero multimer comprises heterodimer (as Fab, Fc fragment and bivalent antibody), tripolymer G-protein, Both heterotetrameric (as F (ab ') 2 fragment and IgG) and also has oligomer structure.

" phagemid " or " phage vector " is containing being derived from the two the rf cloning vector of component of phage chromosomal and foreign DNA (as from those of plasmid).Because phagemid contains a part for phage genome, with helper phage simultaneously infection host time, it can be packaged into phage particle.Phagemid of the present invention can be packaged into phage M13 particle.

What term " surface of phage particle " referred to phage particle is contained in the part of medium contact wherein with this phage particle.The surface of phage particle is assembled by coat protein and is determined (member of the assembling of the protein enclosure of phage particle), the internal surface of coat protein assembling has a common boundary with the region of the nucleic acid containing phage, and wherein said nucleic acid copies in phage manufacturing processed in suitable host cell.

So-called " bacteriophage coat protein " its mean those protein of the phage ghost forming naturally occurring phage.In filobactivirus, as fl, fd and M13, coat protein is gene III protein (pIII), gene VI albumen (pVI), gene VII albumen (pVII), gene VIII protein (pVIII) and gene IX albumen (pIX).Difference between the sequence of the coat protein of M13 and the closely-related member of filobactivirus is well-known to those skilled in the art (see such as Kay, B.K., Winter, J. & McCafferty, J. edit, (1996) .Phage display of peptides and proteins:a laboratory manual.Academic Press, Inc., San Diego).

general introduction

When producing the fusion rotein formed with bacteriophage coat protein, comparatively greatly or different chemical propertys can be had relative to bacteriophage coat protein foreign protein molecule, particularly like this for pIX or pVII, the assembling of recombinant phage particle may be disturbed.In order to avoid assembling interference, often use phagemid system, wherein phage particle assembles when there is helper phage also thus not only containing expressed wild-type protein but also containing foreign protein-coat protein fusions.Because need extra step phagemid system comparatively loaded down with trivial details, and the complete complementary much lower (if any) of the coat protein of showing foreign protein can be caused.Because the event of a rear problem, phagemid display systems is considered as being unit price usually.On the other hand, phage vector system being used for realize multivalent display often causes the phage of low titre to grow.

The present invention is based on such discovery: first base from the codon of single nucleotide mutation namely the 6th of this sequence the residue in the encoding sequence of the pectin lyase B secretion signal (pelB) of carrot soft rot Erwinia sports T by C, proline(Pro) in coded signal (SEQ ID NO:1) is caused to change to Serine, the improvement that some albumen (being included in the protein that M13 bacteriophage coat protein is shown) can be caused to secrete from host bacterium film is realize to the secretion in pericentral siphon district by improving peptides and proteins by inference.Secondly, the sudden change (the A11P variant of SEQ ID NO:3) of ompA secretion signal has shown the titre of the phage that can strengthen displayed polypeptide-coat protein fusions.

Use P6S pelB variant secretion signal, applicant is verified, utilize phagemid system, comprise a large amount of positive charge of band, with a large amount of negative charge or the peptide of peptide of disulfide linkage constraint can successfully show on pIX and pVII bacteriophage coat protein, and antibody fragment and closer time, homodimeric antibody structure, comprises the fusion rotein that total length IgG successfully can be shown as pIX coat protein.In addition, example as shown here, utilize P6S pelB and ompA, use the various different peptide of phage vector as herein described and small protein matter structural domain multivalent display to be the fusions formed with pVII.That to have used saltant type pelB secretion signal described herein (SEQ ID NO:1) to carry out large and that polymeric protein is on pIX success and effectively showing.Protein as herein described on pVII and pIX while multivalent display have employed pelB secretion signal (SEQ ID NO:1) and the ompA secretion signal (SEQ ID NO:3) of saltant type, but, can by this pelB secretion signal or this ompA secretion signal with treat that both the protein of multivalent display and bacteriophage coat protein fusions are combined.

These results show, comprise found modification pelB signal sequence and simplify for utilizing phagemid vector or phage vector to show in pIX or pVII coat protein, the system of foreign protein can be used for showing biochemical diversity peptides and proteins.In addition, this system is utilized also can to show to comprise beaded support (scaffold) as other albumen of the Z structural domain of the fibronectin domain of fibronectin, connetin etc., albumin A, ankyrin repetition, PDZ structural domain, Knottin, CTLA-4 ectodomain.Monomer, dimeric or polymeric receptor protein is as those (as erythropoietin receptor and the acceptors of ERBB family comprising heregulin) of being combined with somatomedin, neurotransmitter receptor (as γ-aminobutyric acid) and other organic or inorganic small molecule receptor are (as mineralocorticoid, glucocorticosteroid) be the example that available code is selected from the foreign protein of the carrier displaying of the saltant type secretion signal of SEQ ID NO:1 and 3, described secretion signal and polypeptide chain (comprise wild-type, saltant type or truncation type receptor polypeptides chain) encoding sequence connect.Preferred Heterodimeric receptor body is the nuclear hormone receptor (people such as Belshaw, (1996) Proc.Natl.Acad.Sci.U.S.A 93 (10): 4604-4607), erbB3 and erbB2 receptor complex, and g protein coupled receptor (includes but not limited to opiate receptor (people such as Gomes, (2000) J Neuroscience 20 (22): RC110); The people such as Jordan (1999) Nature 399:697-700), the acceptor of M-ChR, Dopamine Receptors, serotonin receptor, Adenosine Receptors and GABAB family).In addition, use randomization and diversified standard technique, the structure for the display carrier of above-mentioned monomer, dimer or polymer protein can be used as the basis in the library being structured in the Variant molecules that phage is shown.This library can be for exploring reinventing of protein and again engineered useful tool according to these motifs and support.

Antibody component as herein described is successfully shown as the fusion rotein formed with pIX or pVII coat protein by applicant of the present invention unexpectedly on the surface of filobactivirus, its homodimeric body protein connected as disulfide linkage, the at least one known organism demonstrating the Fc structural domain of natural antibody is active, as Fc receptors bind, and when form protein-bonded for bivalent antigen, can with target antigen specific binding.The ability expressing the large multi-domain proteins be connected with phage protein is not also reported for work before this, and this ability it is believed that just or part is owing to using saltant type peIB secretion signal in foreign protein construct.

implement method of the present invention

In the fusion rotein that filamentous phage particle is shown, " fusion " between allogenic polypeptide and filamentous phage coat protein (and particularly pVII or pIX albumen) can be directly be connected by amido linkage, or can comprise connecting peptides (i.e. " joint ").Can use any one in multiple joint, it is about 5 to 50 amino acid whose sequences that described joint is generally a segment length.Especially preferred joint can provide the reactivity of height to fusion rotein in joint.Lack the joint of secondary structure, as primarily of glycine (G, Gly) residue form those, to repeat or those (number wherein repeated is generally 1 to 12) of G3S (Gly-Gly-Gly-Ser) can be used for this object as having G4S (Gly-Gly-Gly-Gly-Ser).

First polypeptide is exogenous protein, the second polypeptide be filamentous phage coat protein as pVII or pIX albumen, wherein this exogenous protein merges to the N-terminal of this filamentous phages body protein.In addition, when fusion rotein is prematurity form, namely wherein homing sequence also not processed (removing), fusion rotein can containing the prokaryotic secretion signal variant of aminoterminal pelB or ompA as described herein.

If expect to express antibody (immunoglobulin (Ig)) molecule, the polynucleotide molecule of this immunoglobulin (Ig) of then encoding will comprise: (1) first cistron, comprise and the Translation initiator of the polynucleotide sequence effective integration of encoding heavy chain and secretion signal and (2) second cistrons, comprise and the Translation initiator of the polynucleotide sequence effective integration of coding light chain and secretion signal, when wherein expressing this light and heavy polypeptide chain in prokaryotic organism host cell, this light chain and heavy chain fold and assemble and form biological activity immunoglobulin (Ig).Formed being desirably in the surface expression antibody of phage or can the stablizing associating of antibody dimer or Heterodimeric body structure at least partially time, one or both in described cistron is by encoding fusion protein, described fusion rotein comprises the saltant type secretion signal of the present invention being selected from SEQ ID NO:1 and 3 merged with immunoglobulin domains, and itself and bacteriophage coat protein merge.

In natural antibody, light chain polypeptide chain and heavy chain polypeptide chain are encoded independently and express.The typical Heterodimeric body structure of IgG quasi-molecule depends on correct assembling between four polypeptide chains (two heavy chain and two light chains) of this molecule and disulfide formation.Thus, in the present invention, the assembling of dimerization Fc part of antibody and/or the associating (when existing) of light chain have reappeared the natural process that antibody is formed, and wherein each structural domain oneself connection of protein is incorporated between the two and forms disulfide linkage.

In one embodiment, to be natural antibody containing Fc protein at the displaying on surface of filamentous phage particle, and construct for Fc construct-pIX fusion rotein or Fc construct-pVII fusion rotein and will oneself absolute coding of associating and the bi-cistronic vectors of the antibody Lc of expression or the expression of antigen-binding domains.Antigen-binding proteins of the present invention can have the binding site of any epi-position, antigen site or protein.Preferred antigen-binding proteins by directly with receptors bind or by the combination of the cognate ligand with them and neutralization, in coming and receptor protein activate.In general, antigen-binding domains is formed by the antibody Lc merged with the natural antibody Hc sequence comprising Fc structural domain and antibody Hc variable domains.In another embodiment, coat protein-fusion rotein comprises the scFv be connected with Fc structural domain.In another aspect of the present invention, form the heavy chain of scFv and the antigen binding site of light chain and can change to provide two kinds of different binding specificities, thus make the construct protein connected at the disulfide linkage of the self-assembly of phage display be the bivalent molecule of dual specific.Such as, the V of IgG molecule is substituted _land V _hstructural domain be that there is not homospecific scFv structural domain, make the molecule of gained can simultaneously in conjunction with two kinds of different epi-positions.Produce the additive method with the right bi-specific antibody molecule of multiple variable domains and have instruction in US20020103345A1, these bi-specific antibody molecules can be shown on phage particle by method of the present invention, the document are incorporated to by reference herein.

In one embodiment, antigen combine or receptor binding domain be not come from antibody domain but with the known or random peptide sequence of Fc domain fusion.Application WO04/002417, WO04/002424, WO 05/081687 of the common pending trial of applicant and WO05/032460 describes and is referred to herein as MIMETIBODY ^tMthe structure of structure, the each of these patent applications is incorporated to herein by reference in full, included described structure the dimeric structure connected as disulfide linkage, and it can melt with pIX or pVII bacteriophage coat protein the outside surface being incorporated in phage particle shows.

In one embodiment, the protein that phage particle is shown comprises a pair biologically active peptides-joint-hinge-CH2-CH3 polypeptide, and this is to polypeptide by associating or covalent linkage, and particularly Cys-Cys disulfide linkage connects.Biologically active peptides can have any length and can be derived from any species naturally occurring sequence or for artificial sequence.This peptide usually will be encoded by phagemid vector and with the Fc meromixis of the construct for showing on phage particle.Other constructs with similar structures can with the vector expression being mixed with saltant type secretion signal of the present invention, wherein said peptide has unknown activity but it shows the effect playing marker, label, antigen, or provides puting together of reporter group, chelation group etc.

The expression level of secreted protein or coat protein-fusion rotein also can be controlled in addition on transcriptional level.Fusion rotein be in Lac Z promotor/operon system induction type control under (see Fig. 1).Other inducible promoters also can use and be well known by persons skilled in the art.In order to high-level surface expression, sublibrary (suppressor library) will be suppressed at the inductor of Lac Z promotor as cultivated in isopropylthio-beta galactose glycosides (IPTG).It is favourable that induction type controls, because quite the bioselection of non-functional pIX fusion rotein comes minimized by cultivating this library under non-express condition.Then can only screen time abduction delivering to guarantee that in library, whole antibody population is accurately presented in phage surface.

The carrier of the protein secreted by coding or coat protein-fusion rotein can comprise translation stop codon at the joint of foreign protein coding region and bacteriophage coat protein coding region.When expressing in the bacterial cell carrying corresponding Transcription Termination suppression, this fusion rotein is produced.When expressing in the bacterial cell without corresponding suppression, free foreign protein can not produce.

use method of the present invention

When saltant type secretory signal sequence of the present invention is used for the carrier building the protein expression be used in bacterial host cell (as intestinal bacteria), polypeptide secreted enter host cell pericentral siphon in and it is therefrom reclaimed.Protein recovery is usually directed to destroy microorganisms, is generally destroyed by the means of such as osmotic shock, supersound process or molten born of the same parents and so on.Once cell is destroyed, namely remove cell debris or intact cell by centrifugal or filtration.Can be further purified protein, such as, by affine resin chromatography purifying.Or, protein transduction can be transported in substratum and from wherein isolated protein.Cell can be removed from culture, culture supernatants be filtered and concentrates the protein be used for produced and be further purified.Available usually known method such as polyacrylamide gel electrophoresis (PAGE) and protein imprinted assay method carry out further Isolation and Identification to expressed polypeptide.

When saltant type secretion signal of the present invention being used for structure and being used for phage vector or the phagemid vector of the expression of fusion rotein (it is the foreign protein merged with bacteriophage coat protein), described protein is the surface-mounted of formed phage particle and secrete from host cell membrane (as Bacillus coli cells).

Phage vector exemplified here is used as starting point, site-directed mutagenesis can be utilized to carry out variation process to produce molecular library at specific discrete resi-dues or in the region of such as N-connection glycosylation sequences (being commonly referred to NXT sequence) and so on to protein or foreign protein-bacteriophage coat protein fusions.Useful especially is the Kunkel mutafacient system improved, and the method can be used for generation hundreds of millions E. coli clones, and each bacterium colony has different foreign protein sequences.Although effectively, when producing the Sequence Library of high complexity, the per-cent of non-mutagenic parent DNA increases.In addition, when for the preparation of when comprising the library of sequence polymorphism compared with far region, the technical limitation of long oligonucleotide synthesis can reduce the validity of the method.In order to overcome these restrictions, other generation can be used more than the technology of the oligonucleotide of 350 bases.These technology comprise the Kunkel mutafacient system (people such as Kunkel using huge primer and produce the neck-ring sequence combined standard containing Restriction Enzyme recognition site in mutagenesis template, 1987 Methods Enzymol.154:367-382), as described in US20050048617.With other library technology as restriction enzyme digestion clones (restriction cloning) (people such as Marks, 1991 J.Mol.Biol.222:581-597; The people such as Griffiths, 1994 EMBO J.13,3245-3260; The people such as Hoet, 2005 Nature Biotechnol 23,344-348), phage restructuring (Gigapack, Invitrogen) compare with sequence-specific restructuring, the method based on Kunkel of this improvement in generation sequence polymorphism library (being greater than 109) obviously more effectively and any position calling sequence diversity be easier at target DNA.

When expecting to screen large this molecular population for required binding characteristic, particularly useful containing the displaying of Fc protein on filobactivirus.The bacterial cell of expressing desired protein or protein fusions is infected with the M13 variant allowing the carrier DNA carrying fusion gene to be preferentially packaged into phage particle.The phage particle of each gained can show the fusion rotein of one or more copy and the carrier containing this fusion rotein of coding.By the colony of biopanning procedure for required this phage particle of binding characteristic enrichment.Usually, required particle is fixed on the solid surface (as elisa plate) being coated with the antigen that required phage particle can combine with it.Collect combine particle and for further bacterial infection cell.Repeat this biopanning procedure to carry out enrichment further for required binding characteristic.

A kind of method of screening the protein (as antibody or the molecule containing Fc) reclaimed from phage library removes the bacteriophage coat protein for showing being connected to this protein molecule.For the ease of removing bacteriophage coat protein, restriction enzyme site of can encoding between foreign protein and coat protein is also suitably located to comprise or to get rid of label (as Flag or hexahis) sequence.

Phage and other antibody display methods are that the external selection handling relative antigen or receptor targets provides chance.A specific advantage of external system of selection can handle select procedure to obtain the antibody that the multiple site in target protein is combined.Alternatively, intact cell can be used for selecting binding substances.

Phage library simplifies the retrieval to the genetic stocks relevant to functional attributes, but, need the elutriation strategy of multi-step to come to be separated best material standed for from library.The elutriation that structural domain or epi-position guide has become the usual manner selecting the antibody be combined with target protein.This selection being called selectivity elutriation (selective panning) mainly through utilizing, removing the method selecting elutriation (de-selective panning), part trapping (ligand capture), subtraction elutriation (subtractive panning) or pathfinder selection (pathfinder selection), adopts the substep of antagonist to select to realize.

In subtraction elutriation, can be used for removing the selection to less desirable binding substances by having the overlapping but target in incomplete same site.Be used for this strategy differentiating the binding substances of even unknown antigen, as use normal cell make a return journey the binding substances selecting cancer cells usage.Alternatively, the naturally occurring protein with some common structural domain or structure is used for select progressively or tournament selection to obtain different from related antigen or that identical site combines antibody.In some cases, the mutant form of naturally occurring protein as relevant chemokine or protein can be used for subtraction elutriation.

The elutriation that part trapping guides is with similar with ELISA sandwich assay in following: the immobilized antibody for incoherent or non-conterminous epi-position be used for the preferred bonding surface of dump target part and be provided for phage elutriation (US6376170).Someone has used competitive antibody optionally to cover antigen (people such as Tsui, P., 2002.J.Immunol.Meth.263:123-132) at non-required targeting domains place.Pathfinder selection technology uses monoclonal antibody and polyclonal antibody, and directly or indirectly and the native ligand puted together of horseradish peroxidase (HRP).When there is vitamin H tyrasamine, the biotinylation of the phage that these Journal of Molecular Catalysis next-door neighbour target antigen combines, thus make to reclaim the phage of " tape label " by Streptavidin specificity from total entirety.In like fashion, combine with target itself, or the phage that next-door neighbour target place combines is able to selective recovery people such as (, 1998.Immunotechnol.3:293-302) Osborn, J.K..The variations of these methods, these methods and additive method well known by persons skilled in the art can be adopted to inquire about the library of the foreign protein produced by the expression method depending on saltant type secretion signal of the present invention.

In one embodiment, phage library is used for for Fc acceptor that is natural or that recombinate, as the enhancing of the combination of FcR γ III (CD16), FcR γ II (CD32) and FcR γ I (CD64), reduction or change, screen the variant of the Fc part of molecule.

Although briefly describe the present invention, embodiments of the invention also will be open further in the following examples, and described example should not be construed as the restriction to the scope that claim is protected.

the multivalence peptide of example 1. on pVII is shown

Whether effectively and stably can show the different peptides and proteins of multiple copied in order to test pVII, designing and constructing phage genome display carrier.PVII carrier phage genome M13KE builds, and M13KE is the derivative of M13mp19 (be the carrier of the derivative of strand, male specificity thread DNA phage M13, NCBI NC_003287, grow up about 7250bp).PVII and pIX encoding sequence is adjacent one another are in genome.In this carrier, the peptide with initiator codon and GSGGG joint is inserted in the N end of pVII (Figure 1A).The BspH I site of the BsrG I restriction endonuclease sites before pVII coding region and the end in pIX coding region is used for clone.

A series of biochemical diversity peptide (comprising linear peptides (HA), peptide (Flag) with a large amount of negative charge, the peptide (6 is histidine-tagged) of positively charged and the peptide (PHPEP 190, PHPEP 97) of disulfide linkage constraint) to be cloned in this carrier and to carry out testing (table 1).By over-lap PCR produce coded signal sequence, peptide, GS joint, pVII and pIX DNA fragmentation and extend 5 ' and 3 ' side DNA sequence dna on end of BsrG I and BspH I recognition site.Digesting the PCR primer through gel-purified by BsrG I and BspH I restriction endonuclease, then connecting the double-strand M13KE phage into digesting by same enzyme.The recombinant phage dna of connection is transformed in XL1-Blue competent escherichia coli cell (Stratagene).

the peptide sequence that table 1.pVII shows

SEQ ID NO：	Peptide title	Sequence
			5	HA	YPYDVPDYA
6	FLAG	DYKDDDDK
			7	6xHis	HHHHHH
8	PHPEP 190	GGDPCTWEVWGRECLQGG
			9	PHPEP 97	RECHPQNWTSCSN

Then to be separated independent plaque on the lawn cell of conversion being inoculated in XL1-Blue host cell.By the plaque settling flux on XL1-Blue E. coli lawns in phosphate buffered saline (PBS) (PBS) to allow phage particle diffuse out from top-agar.The phage diffused out is added into XL1-Blue liquid nutrient medium and is cultured to O.D.0.5 and carry out ehec infection cell.Culture is shaken 5 hours at 37 DEG C.After concussion by centrifugal except degerming.By adding the cold 0.5M sodium-chlor of 1/10th volumes, 4%PEG-800 this mixture incubation at 4 DEG C is precipitated the phage particle in supernatant liquor for 3 hours.Then centrifugal mixture by phages thing settling flux in PBS, Aliquoting is also preserved at-20 DEG C.

Measure the combination (Phage-ELISA) of immobilized part on complete phage and microtiter plate, carry out DNA sequencing and titration is carried out to phage titre, characterize phage and evaluate their displayings on phage surface.Antibody or the protein bag of the peptide shown with the identification that 100 μ L/ hole concentration are 5ug/mL (being buffered in liquid pH 9.5 at carbonate bag) by black Maxisorp elisa plate (NUNC) are spent the night.With the TBS (TBST) containing 0.1%Tween-20, the plate of bag quilt is washed 3 times, at room temperature close 1 hour with Chemiblocker (Chemicon International).The Phage samples diluted in Chemiblocker is added into the plate for combining.Incubation at room temperature, after 1 hour, washs this plate again to remove unconjugated phage.Detect antibody (anti-M13 antibody/horseradish peroxidase conjugate (GE healthcare)) 1: 5,000 to be diluted in TBST/10%Chemiblocker and to be added into this plate with 100ul/ hole, incubation at room temperature 1 hour.After final washing, in order to detect, POD chemical luminous substrate (Roche) is added into this plate with 100 μ l/ holes, read plate instrument to carry out read plate at Tecan at once.

result

This Phage-ELISA data have been shown in Fig. 2 A-E, although confirm to have different structure and bio-physical property, whole five kinds of peptides all successful presentation on phage surface, are connected with pVII coat protein.

What illustrated in Fig. 3 that phage shows with phagemid compares, wherein pentavalent is shown on bacterial body system (pVII of whole five copies all shows fusion rotein) and pIX from hybridizing display systems (phage vector that is that there is wild-type and both pIX encoding sequences that are that merge, p99, as described in WO2009/085464 and WO2009/085468) result compare, in this hybridization display systems, wherein the peptide of an average only copy is demonstrated.ELISA result confirms, is obviously better than being derived from the phage of p99 crossing system, thus shows that this carrier system creates multivalent bacteriophage display in the combination being derived from HA peptide and the anti-HA antibody that the phage of pVII phage vector is shown.

the impact that example 2. secretion signal is shown peptide

Wild-type pVII is the protein not having signal peptide of natural synthesis, although it is inner membrane protein in host cell.But, in the former research to pVII and pIX display systems, employ signal peptide (people such as Gao, 1999, Proc Natl Acad Sci U S A 96:6025; The people such as Kwasnikowski, 2005 Journal of Immunological Methods 307:135-143).Whether in order to check bacterium signal sequence necessary to the phage display on pVII, we create the construct wherein adding or do not add pelB at HA peptide (being thereafter GSGGG joint and pVII) above.

Fig. 4 is presented at HA when not having pelB can be shown on phage surface.But, indicated by more weak ELISA signal, HA shows, even if the number of phage particle used identical with the number of the phage with pelB (Fig. 4) with obvious lower efficiency on phage surface.ELISA data show, although signal sequence pelB not necessarily, improve peptide and show, are by inference to be undertaken realizing in the film assembled by helping to bring the pVII peptide that HA merges into phage.To the time-histories research instruction of phage growth, the phage being derived from the phage vector with pelB is faster than the phage growth being derived from the phage vector without pelB, thus produces evidence further to show that bacterial signal peptide contributes to the assembling of the phage that HA shows from the teeth outwards.

example 3:pelB suddenlys change

PHPEP 97 (RECHPQNWTSCSN) is 13 the amino acid whose peptides retrained by disulfide linkage be combined with Streptavidin, is one of the peptide of test pVII phage display (example 1).In Phage-ELISA, two PEP97 clone (CL#7 and CL#8) displays increase (Fig. 2 E) with the binding ability of Streptavidin.Carry out sequential analysis to these two clones and found that the single amino acids in these two pelB sequences of cloning suddenlys change, the 6th proline(Pro) sports Serine (Fig. 5 A).

Then the ability strengthening the displaying of other peptides for P6S pelB is tested it, also observes the improvement (data are not shown) showing efficiency.Whether affect protein secreting to test this sudden change, the pelB of pelB or sudden change and alkaline phosphatase (AP) are merged and examine the secretion of AP in DH10B bacterium.

Have detected alkaline phosphatase activities secreted in cell culture.Result display (Fig. 6 A), this sudden change significantly improves the secretion of AP to cell culture medium (supernatant liquor fraction) really, but intracellular AP concentration identical (Fig. 6 B).Thus we suppose, by this sudden change, the pelB of sudden change improves PHPEP97-pVII carries out transhipment from the plasma membrane assembled to phage.As far as our knowledge goes, other is not also had about the report of the sudden change that can increase in the peIB of protein secreting.

example 4: the two multivalent display of peptide on phage pVII and pIX

In order to while multivalent display two kinds different peptide, phage vector is constructed based on phage genome M13KE, wherein saltant type P6S bacterial signal peptide pelB (SEQ ID NO:2, C16T), peptide A and flexible GSGGG joint are inserted into the N end of pVII gene; Wild-type bacterium signal peptide ompA (SEQ ID NO:4, G31), peptide B and flexible GSGGG joint are inserted into N end (Figure 1B) of pIX.The BspH I site of the BsrG I restriction endonuclease sites before pVII coding region and the end in pIX coding region is used for clone.By over-lap PCR generation signal sequence, peptide, GS joint, pVII, pIX gene and 5 ' and the 3 ' side DNA sequence dna held extending BsrG I and BspH I recognition site.Digesting the PCR primer through gel-purified by BsrG I and BspH I restriction endonuclease, then connecting the double-strand M13KE phage into digesting by same enzyme.As previously mentioned the recombinant phage dna of connection is transformed into XL1-Blue intestinal bacteria to experience in cell (Stratagene) and the cell of conversion to be inoculated on the lawn of XL1-Blue host cell to be separated single plaque.

Be have rated the displaying (Fig. 7) of both FLAG and HA peptides by Phage-ELISA, this ELISA confirms that two kinds of peptides are all shown on phage particle.For PHPEP 97 displaying too, but initial plaque size is very little, and phage titre is low.

In order to select the spontaneous change that can improve growth velocity in phage, we carry out continuous passage to the phage carrying out two independent clonings from the beginning.After taking turns continuous passage five, the phage titre observing a clone (#11) along with time lapse significantly increases (Fig. 8).The sequence of taking turns the full phage genome of the phage purifying of taking turns with the 10th from the 5th compared, determine an only Single amino acid mutations, be positioned at position 11 place of signal peptide ompA, Ala sports Pro (Fig. 5 B).Also identical sudden change is identified from the phage of other clones (#9).But this sudden change does not have the secretion (Fig. 6 A) of remarkably influenced alkaline phosphatase as the sudden change in peIB.

the displaying of example 5: total length IgG on phage particle

a. carrier design.

From the phagemid construct (pCNTO Fab IX) such as described in WO2009/085462, construct total length IgG show phagemid (vDR47, Fig. 9).With the addition of encoding human IgG1 hinge, the sequence of CH2 and CH3 structural domain and P6S pelB variant signal sequence, this carrier does not have lacI gene, but controls to express by lac promotor.

b. for the sign of the construct of total length IgG displaying.

Prepare a series of test builds body to assess the displaying of total length IgG on pIX.Select to build new total length IgG molecule for the antibody (called after 6-2 and 16-7) of IL13 and anti-cytokine antibodies 9-4 as prototype.In order to determine the effect that different codon is selected, for each in anti-IL13 antibody prepares two constructs, one to have carried out people codon optimized, and one has been carried out e. coli codon optimization.Table 1 lists the container name of five total length IgG test builds bodies.As United States Patent (USP) 6,670,127 and 6,521, the gene of synthesis optimizing described in 427 is also assembled into double-stranded DNA.In addition, the EMP-1 peptide-Fc fusion constructs (the SEQ ID NO:88 of CNTO530, US7393662) merged with pIX is included in contrast, because it contains human IgG hinge, CH2 and CH3 structural domain but without light chain.

the test builds body that table 2. is shown for total length IgG

pDR#	Isotype	Codon is selected	Explanation	Antigen-specific
					pDR2129	huIgG1/HuKappa	People's codon	6-2 total length IgG	People IL13
pDR2130	huIgG1/HuKappa	People's codon	16-7 total length IgG	People IL13

pDR2131	huIgG1/HuKappa	E. coli codon	6-2 total length IgG	People IL13
					pDR2132	huIgG1/HuKappa	E. coli codon	16-7 total length IgG	People IL13
pDR3041	huIgG1/HuKappa	People's codon	9-4 total length IgG	People IL17A

c. phage preparation

The total length IgG described above in " trifle B " according to standard schedule shows that construct is transformed in two different F ' coli strain TG-1 and XL-1blue.The reason of testing these two bacterial strains is their growth velocity differences, and this can affect packaging and the displaying of total length IgG pIX fusion rotein by inference.Choose independent transformant and in the 2XYT substratum being supplemented with Pyocianil (always using with 100 μ g/ml) overnight incubation.Then by this overnight culture (500 μ l) for inoculating 25ml2XYT/ Pyocianil, by culture 37 DEG C, cultivate until OD (600nm) reaches 0.5 under 250rpm.Between the incubation period of 30 minutes, (37 DEG C, nonoscillatory) use 10^ ¹¹the VCSM13 helper phage (Stratagene, La Jolla, CA) of pfu/ml infects this bacterium, then under 3,000rpm, carries out centrifugation step 15 minutes.In this step, standard schedule requirement 2XYT/ Pyocianil/IPTG (1mM) induces this bacterial cultures.But culture is divided into two parts by us, 1mM IPTG is added into portion, another part is not added, and supposes that this system leak will be enough to produce the fusion rotein of the phage packaging had subsequently.Put it briefly, for each construct, prepared four parts of different bacteriophage preparation: (i) TG-1, use IPTG (ii) TG-1, do not use IPTG (iii) XL-1blue, use IPTG (iv) XL-1blue, do not use IPTG.By this culture at 30 DEG C with 250rpm overnight incubation, second day, with 3,000rpm centrifugal 15 minutes, then precipitating phage supernatant liquor in PEG/NaCl.On ice after 2 hours, by the phage of precipitation under 10,000rpm centrifugal 15 minutes, by phages thing settling flux in 2ml PBS.By with centrifugal 10 minutes of 10,000rpm bacteriophage preparation being purified further the bacterial precipitation thing that removes any remnants and being stored in 2ml test tube at 4 DEG C.

d. phage titre

Phage titre is measured according to standard schedule.In brief, TG-1 cell is cultivated in 2XYT until OD (600nm) reaches 0.5.Bacteriophage preparation is carried out serial dilution with PBS in 96 orifice plates, TG-1 cell is added into this phage and at 37 DEG C incubation to infect.After 30 minutes, carry out a titration by being dispensed to by every hole 2ul on the LB agar plate containing 1% glucose and Pyocianil.Be incubated overnight at 37 DEG C by plate, measure the concentration of phage, unit is colony forming unit (cfu)/ml.Table 3 shows the result of the phage titration for all constructs and culture condition.All clones all produce high phage titre, at 10^ ¹¹-10^ ¹³between cfu/ml, this is estimated scope and shows that phage is able to effective preparation.

table 3.

e. in order to the IgG structural domain specific sandwich ELISA of evaluation function displaying

In order to assess the displaying of total length IgG molecule on phage pIX, establish a series of sandwich ELISA.With the one bag in the following trapping antibody diluted in TBS of 1 μ g/ml by black maxisorp plate: goat anti-human IgG (Fd, CH1) antibody (The Binding Site, Birmingham, UK), mouse anti human k light chain (Southern Biotech, Birmingham, AL), mouse anti human IgG (CH2 structural domain) antibody (AbD Serotec, Raleigh, NC) and mouse anti human IgG (CH3 structural domain) antibody (AbD Serotec).With Chemiblocker (Chemicon/Millipore, Billerica, MA), after carrying out closing to plate, wash plate also adds phage and incubation one hour with the concentration of 2 × 10^11cfu/ml (diluting in 10%chemiblocker/TBST).Wash plate the little mouse-anti M13 antibody puted together by HRP is added into plate.Incubation is after 30 minutes, and chemical luminous substrate is also added in hole by wash plate, reads to carry out in plate instrument reading plate at Envision.Figure 10 A-D respectively illustrates the result from CH1 (Figure 10 A), k (Figure 10 B), CH2 (Figure 10 C) and CH3 (Figure 10 D) sandwich ELISA.Contrast for ELISA is that the Fab-pIX fusions of the clone 6-2 shown in vDR10 is (codon optimized through people, prepare in TG-1 cell, induce with IPTG) phage of (for a kind of non-specific scaffolding protein-pIX fusions) or CNTO530-pIX fusions.In CH1 and k ELISA, 6-2Fab is used as positive control, and EMP-1 construct (CNTO530) molecule is used as negative control.In CH2 and CH3ELISA, 6-2Fab is used as negative control, and CNTO530 molecule is used as positive control.This scaffolding protein phage is used as negative control in all ELISA, because it carries any antibody domain.Also add anti-IL13 total length IgG1 antibody using the concentration of 5ug/ml and carry out ELISA mensuration to prevent phage and different trapping antibodies as solubility competitor.

As shown in Figure 10 A-D, phage can be detected in all sandwich ELISAs, thus produce evidence to show that described phage in fact shows different antibody domain from the teeth outwards.The phage produced in XL-1blue cell has the highest signal, and adds IPTG and have positively effect to binding signal.By adding the combination of solubility anti-IL13 antibody suppression phage, this can indicate specificity to interact.But the anti-IL13 antibody of solubility can not compete the interaction (Figure 10 D) between phage and CH3 structural domain.For total length IgG-pIX fusions and for EMP-1-Fc-pIX fusions (CNTO530), the two all observes this point.

f. total length IgG pIX phage is combined with IL13

After confirming to detect on phage particle by ELISA all structural domains of IgG molecule, be necessary to determine whether described construct also retains the ability be combined with they respective antigen.By being set up IL13 in conjunction with ELISA with the commercially available anti-IL13 antibody of 1 μ g/ml (mouse anti human IL13, MAB213, R & D Systems) bag by black Maxisorp plate.MAB213 does not compete the combination with IL13 with 6-2 or 16-7, thus it is desirable as sandwich ELISA trapping antibody.In washing with after closing, add biotinylated people IL13R130Q human (Peprotech) with 100nM and incubation one hour.Wash plate with 2 × 10 ¹¹cfu/ml is added on the phage of 6-2 and 16-7 pIX showing total length IgG form, and this interpolation adds separately or adds together with the anti-IL13 antibody of the solubility for competing.With the phage that the little mouse-anti M13 antibody test that HRP puts together combines, in Envision instrument, read chemoluminescence.Figure 11 shows the result of IL13 Phage-ELISA.Under much said conditions, detect combination, the phage wherein using 1mM IPTG to produce in XL-1blue cell demonstrates the highest signal.Peptide-Fc-pIX and alternative scaffold molecule-pIX fusions are negative (as expected), and 6-2Fab pIX contrasts as positive.Add the combination of solubility anti-IL13 antibody suppression, thus to show this combination be specific.In order to check IL13 to combine further, establishing ELISA, wherein solubility being competed antibody and carrying out serial dilution from 50 μ g/ml-0.01 μ g/ml.Further comprises control antibodies.Figure 12 A and B respectively illustrates the impact that soluble antibody combines the IL13 of 6-2IgG pIX and 6-2Fab pIX.These two constructs all be observed to the suppression of combination, IC50 is about 0.1 μ g/ml.But, for total length IgG pIX construct, even this suppression is incomplete under very high competitor concentration, thus show the non-specific interaction that there is certain level.

g. total length IgG pIX phage is combined with IL13 and IL17

Carry out second authenticity experiment.This experiment is carried out by the anti-IL17A antibody of Cloning of full length IgG form.As mentioned above this construct to be transformed in XL-1blue cell and to prepare phage.Carry out ELISA to confirm the combination of the displaying of IL17IgG on pIX and itself and people IL 17Amut6 antigen, as shown in Figure 13.For each ELISA (Fd trapping, k trapping, CH2 trapping, CH3 trapping, IL13 trapping and IL17 trapping), add separately phage or it is added together with solubility anti-il 13 antibodies or the anti-IL17A monoclonal antibody of solubility.Add the specificity that competitive monoclonal antibody can show ELISA.As what can find out in Figure 12 A and B above, IL17IgG shows on pIX, but level is lower than IL13IgG.This and Fab expression level different (data do not show) between these constructs are consistent.Can find out the specificity that antigen combines, because the anti-IL13IgG on phage is not combined with IL17, the anti-IL17IgG on phage is not combined with IL13.In addition, by combination that their soluble monoclonal antibody counterpart to suppress in the phage of this two type each.

instance X: the displaying containing Fc protein of merging with pVII

In addition, we prove Fc and MIMETIBODY ^tMalbumen can be shown on phage surface by pVII phagemid system.

Claims (25)

1. coding is used for the polynucleotide of pilot protein matter from the separation of the bacterial secretory signal of bacterium membrane translocation, and described secretion signal is selected from the P6S variant SEQ ID NO:1 of pelB.

2. the polynucleotide of separation according to claim 1, it is effectively connected with the sequence of encoding foreign proteins, and wherein said foreign protein is treated from described bacterium plasma membrane secretion.

3. the polynucleotide of separation according to claim 2, the sequence that wherein said foreign protein is also selected from the bacteriophage coat protein of pIII, pVII, pVIII and pIX with coding is effectively connected, and the fusion rotein wherein so formed is treated to secrete from bacterium plasma membrane.

4. the polynucleotide of separation according to claim 3, wherein said foreign protein is selected from the polypeptide comprising peptide.

5. the polynucleotide of separation according to claim 4, wherein said peptide is heavy chain of antibody constant domain, heavy chain of antibody, mammalian receptors chain, acceptor ectodomain, fibronectin-like domain or ankyrin repeat domains.

6. a carrier, described carrier comprises the nucleotide sequence of encoding bacterial secretion signal be effectively connected with the polynucleotide that come from the aminoacid sequence of exogenous protein of encoding, the secretion signal of described coding is selected from the P6S variant SEQ ID NO:1 of pelB, wherein compared with wild-type secretion signal, described secretion signal strengthens the ratio from bacterium membrane translocation foreign protein out.

7. carrier according to claim 6, the sequence that the exogenous protein encoding sequence of wherein said coding is also selected from the filamentous phage coat protein of pIII, pVII, pVIII and pIX with coding merges.

8. carrier according to claim 7, described carrier is phagemid vector.

9. carrier according to claim 7, the foreign protein encoding sequence be wherein connected with saltant type secretion signal merges with pVII and the foreign protein sequences be connected with saltant type secretion signal and pIX merge, thus causes two kinds of foreign proteins described in when described carrier copies in host cell to appear on the surface of described phage particle with multiple copied.

10. carrier according to claim 7, the foreign protein of wherein said coding is selected from peptide.

11. carriers according to claim 10, wherein said peptide is heavy chain of antibody constant domain, heavy chain of antibody, mammalian receptors chain, acceptor ectodomain, fibronectin-like domain or ankyrin repeat domains.

12. phage vectors according to claim 6, described phage vector also comprises inducible promoter.

13. phage vectors according to claim 12, wherein said inducible promoter is the mutant of lac promotor or lac.

14. 1 kinds of filamentous phage particle, described phage particle is encapsulated with the polynucleotide of the secretion signal that coding according to claim 3 is effectively connected with foreign protein, and on the surface of described phage, have described foreign protein.

15. filamentous phage particle according to claim 14, the amino-terminal fusion of wherein said foreign protein and filobactivirus pVII or pIX albumen, therefore when described protein is when the surface expression of filamentous phages body protein, the cysteine residues on the second foreign protein that the cysteine residues in a foreign protein and the surface of same filamentous phage particle are expressed is oxidized bonding and forms functional protein structure.

16. filamentous phage particle according to claim 15, wherein said functional protein structure comprises antibody Fc district.

17. filamentous phage particle according to claim 16, the functionally active of wherein said protein structure is that FcRn combines.

18. 1 kinds of bacterial host cells, described host cell comprises filobactivirus according to claim 14.

19. 1 kinds of phage libraries comprising the bacterial host cell of Filamentous phage vectors according to claim 18.

20. phage libraries according to claim 19, each polynucleotide encoding wherein said has the sequence of the formation Fc of amino-acid substitution.

21. phage libraries according to claim 20, the sequence of wherein said nucleic acid chains wherein said formation Fc also comprises ligand binding domains.

22. phage libraries according to claim 21, wherein said ligand binding domains is selected from receptor-binding ligands, acceptor ectodomain, the monoclonal antibody structural domain comprising variable domains and constant domain and single chain Fv constructs.

23. phage libraries according to claim 22, wherein said Fab structural domain is included in the sequence that in described binding domains, specific residue place is mutually different.

The polynucleotide that 24. 1 kinds of uses comprise the encoding sequence of the P6S variant of pelB express the method for foreign protein in bacterial host cell, and wherein said foreign protein can reclaim from the substratum of the growth supporting described bacterial host cell.

The polynucleotide that 25. 1 kinds of uses comprise the variant of bacterial secretory signal according to claim 1 express the method for foreign protein in bacterial host cell, wherein said protein and filamentous phage coat protein merge, and in the titre of the phage of protein described in the displaying on surface of described phage particle higher than titre when using wild-type bacterium secretion signal or when not using bacterial secretory signal.